The Overall Goal of this PPG is to develop a novel treatment option for head and neck squamous cell carcinoma (HNSCC) that results in treatment responses that are as effective as current treatment modalities, but that preserve organ function. During the last funding period we demonstrated that HPPH-PDT of HNSCC resulted in complete responses in 82% of patients. In the ancillary studies that accompanied this trial we used noninvasive fluorescence spectroscopy to measure HPPH levels within the lesion. Although preliminary, these studies suggested that non-responding lesions retained low HPPH levels. Simultaneous studies were carried out in this Project using an innovative 3-demensional co-culture system that incorporates patient-derived primary epithelial lung tumor cells and tumor-associated stromal cells from the same patient. The results of this study showed that some tumor cells fail to retain HPPH and were resistant to PDT killing. Subsequent studies showed that HPPH binding to the cell surface, mode of cellular uptake, intracellular localization, subcellular redistribution, exocytosis, and retention was critically dependent upon the structure of the HPPH macrocycle. The long-range goal of this Project is to develop HNSCC-specific photosensitizers that are selectively retained in tumor cells and result in increased efficacy of clinical PDT. To accomplish this goal the following hypotheses will be tested: 1) Photosensitizer (PS) retention characteristics are tumor-dependent and are reflected in the response to HPPH-PDT; 2) Tumor cell properties can be identified that are responsible for PS retention 3) Structural modifications of HPPH can be identified that will enhance PS retention in tumor cells; 3) Targeting of PS to cell surface addresses, such as integrins or epidermal growth factor receptor (EGFR), will further enhance the therapeutic PS differential between non-tumor and tumor epithelial cells. To test these hypotheses we will take advantage of the novel and unique tools developed in the current funding period: a) a compound ?library? of macrocycle-modified HPPH analogs and b) a 3D culture system for primary human HNSCC that has revealed different retention characteristics for individual tumors. The studies proposed in this project are compelling in that they have the potential to enhance our understanding of PS retention and ultimately lead to the generation of HNSCC-specific PS that result in increased efficacy and selectivity of PDT against this disease. The studies are critical to the PPG as they lay the foundation for our long term goal of achieving 100% efficacy in the treatment of early stage HNSCC.